Immunolocalization of synaptotagmin for the study of synapses in the developing antennal lobe of Manduca sexta.

In the mature olfactory systems of most organisms that possess a sense of smell, synapses between olfactory receptor neurons and central neurons occur in specialized neuropil structures called glomeruli. The development of olfactory glomeruli has been studied particularly heavily in the antennal lobe of the moth Manduca sexta. In the current study, we address the development of synapses within the antennal lobe of M. sexta by reporting on the localization of synaptotagmin, a ubiquitous synaptic vesicle protein, throughout development. A cDNA clone coding for M. sexta synaptotagmin was characterized and found to encode a protein that shares 67% amino acid identity with Drosophila synaptotagmin and 56% amino acid identity with human synaptotagmin I. Conservation was especially high in the C2 domains near the C-terminus and very low near the N-terminus. A polyclonal antiserum (MSYT) was raised against the unique N-terminus of M. sexta synaptotagmin, and a monoclonal antibody (DSYT) was raised against the highly conserved C-terminus of D. melanogaster synaptotagmin. In Western blot analyses, both antibodies labeled a 60 kD protein, which very likely corresponds to synaptotagmin. On sections, both antibodies labeled known synaptic neuropils in M. sexta and yielded similar labeling patterns in the developing antennal lobe. In addition, DSYT detected synaptotagmin-like protein in three other insect species examined. Analysis of synaptotagmin labeling at the light microscopic level during development of the antennal lobe of M. sexta confirmed and extended previous electron microscopic studies. Additional synapses in the coarse neuropil and a refinement of synaptic densities in the glomeruli during the last one-third of metamorphic development were revealed.

Enteral absorption of erythropoietin in the suckling rat.

Milk contains biologically relevant concentrations of erythropoietin (Epo), the primary hormone responsible for erythrocyte production. In animals, milk-borne Epo stimulates erythropoiesis. Epo receptors have been found in nonerythropoietic tissues including gastrointestinal tract. We hypothesized that milk-borne Epo is distributed to local gastrointestinal tissues, absorbed intact, and then distributed peripherally via the systemic circulation. Rat milk protected recombinant human Epo (rhEpo) from degradation in the suckling rat gastrointestinal tract. Simulated digestion of (125)I-rhEpo in suckling rat gastrointestinal juices was performed. When measured by acid precipitation and immunoassay, rat milk protected rhEpo from gastrointestinal juices better than saline (p < 0.0001). The fate of enterally administered milk-borne (125)I-rhEpo was examined in 10-d-old rats. RhEpo fed in rat milk was better protected from in vivo proteolytic degradation than rhEpo in saline (p < 0.05). After enteral (125)I-rhEpo dosing, radiolabeled protein from gastric tissue comigrated on SDS-PAGE with intact rhEpo at 36.5 kD. To determine the local and systemic distribution of physiologic intakes of rhEpo, suckling rats were fed (125)I-rhEpo in rat milk, and tissues were harvested 1, 2, and 4 h later. Intact (125)I-rhEpo was found in gastric and small intestinal walls and lumens. Five percent of total administered dose was found intact in the plasma, whereas another 8 to 10% of total administered dose was localized to bone marrow, percentages comparable to those seen after parenteral administration. Radiolabel was also localized to liver and peripheral solid tissues. These patterns of localization and degradation of rhEpo after acute administration support both systemic absorption and gastrointestinal cellular processing.